Sample Preparation Fundamentals for Chromatography - Agilent

364 Pages·2013·7.81 MB·English

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Reliably extract and concentrate
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Table of Contents
Chapter 1 Introduction....................................................................................................1
Sample preparation is an essential part of successful
Chapter 2 Types of Samples and Overview of Approaches to Processing..........9 chromatography. It extends column lifetime, reduces
the need for repeated samples, and minimizes
Chapter 3 General Considerations in Sampling........................................................17
interferences that can jeopardize your separation,
Chapter 4 The Sampling and Sample Handling of Solid Materials.......................20
detection, and quantification.
Chapter 5 Filtration..........................................................................................................29
Chapter 6 Sample Introduction and Sample Preparation Agilent offers the most complete line of sample prep
of Volatile Organic Compounds.................................................................35 products across the full spectrum of instrumentation.
These include:
Chapter 7 Sample Pre-treatment for Liquid Samples..............................................55
• Pre-packaged QuEChERS kits– make sample
Chapter 8 QuEChERS, Salting Out Liquid-Liquid Extraction,
preparation faster, easier, and more reliable.
and Related Techniques..............................................................................78
Chapter 9 Solid Phase Extraction (SPE)......................................................................94 • Captiva Filtration products– improve both system
Chapter 10 Special Topics in SPE...................................................................................133 performance and analytical quality and prevent
extractables, proteins, lipids, or other contaminants
Chapter 11 Size-Exclusion Chromatography
from interfering with the accuracy of your analyses.
as a Sample Preparation Technique.........................................................160
Chapter 12 Column/Flash Chromatography • Chem Elut products– for supported liquid extraction
as a Sample Preparation Technique.........................................................170 (SLE) reduce solvent usage and time over traditional
Chapter 13 Column-Switching (On-Line SPE) liquid-liquid extraction.
as a Sample Preparation Technique.........................................................172
• Bond Elut SPE products– selectively remove
Chapter 14 Sample Preparation Methods for Solid Samples...................................181
interferences and/or analytes from challenging
Chapter 15 Sample Preparation for Biological Samples............................................195 matrices. Choose from the largest selection of
sorbent formats on the market today.
Chapter 16 Sample Preparation in Bioanalysis...........................................................227
Chapter 17 Sample Pre-treatment for LC/MS.............................................................255
www.agilent.com/chem/sampleprep
Chapter 18 Membrane Techniques in Sample Preparation......................................269
Chapter 19 The Role of Scavengers in Sample Preparation.....................................281
Chapter 20 Derivatization for HPLC and GC Analysis................................................286
Chapter 21 Just EnoughSample Preparation:
A Proven Trend in Sample Analysis..........................................................297
Chapter 22 Current Trends and Future Directions in Sample Preparation............307
Chapter 23 Glossary..........................................................................................................310
Index................................................................................................................343
Matrix/Analyte Index...................................................................................351
Preface
Since most samples encountered in a chromatography laboratory are not in a form to be directly placed into the
analytical instrument, some form of preparation is required for nearly every sample. The sample preparation could be
as simple as “dilute and shoot” or as complex as multistage sample handling. The analytical cycle represents all of the
steps from the point of collection to the final analysis and data output. Although sample preparation is an important
part of this analytical cycle, it doesn’t always get the respect as does the separation and measurement instrumentation
and the data handling aspects. Oftentimes, the task of sample preparation employs decades old technology that is
often manual, time-consuming and uses a lot of glassware and other devices, and some older technologies use copious
amounts of solvent that must eventually be disposed of, creating expense and safety issues. Because of the use of
multiple sample preparation steps in an attempt to simplify and/or isolate the desired analytes from a complex matrix,
errors tend to creep into the assay and analyte recoveries may suffer.
The purpose of this book is to outline some of the most popular sample preparation technologies in current use today.
Since sample preparation technologies is represented by tens of possible sample manipulations (e.g. weighing,
dissolution, extraction, trapping, etc.), I didn’t set out to cover every single sample prep category. The book started out
to be a small handbook like the popular The LC Handbook: Guide to LC Columns and Method Development(Publication
Number 5990-7595EN) but soon blossomed into a 350+ page book covering many different sample preparation
technologies. Since the book was written for Agilent Technologies, many of the methodologies covered are those within
Agilent’s chemistries portfolio, but for the sake of completeness, I have covered a number of technologies outside of
Agilent’s immediate areas of interest. Examples of applications are provided throughout the book and many of them
are web-accessible.
Since this book is primarily designed for the chromatography laboratory and to keep the length reasonable, I had to
confine my coverage to organic and biological sample preparation and thus inorganic sample prep, although important
in chromatographic and obviously the spectroscopic analyses of many sample types, was omitted. Many of the techniques
covered, however, such as ion exchange SPE, liquid-liquid extraction and microwave-assisted extraction are equally
applicable to inorganic samples for further analysis by ion chromatography or for spectroscopic measurements.
i
I have written the book in a slightly different format than might be typical. After an introductory Chapter 1 on the
sample prep process, in Chapter 2, I decided to tabulate most of the methodologies that will be covered in the
remainder of the book. That way, the reader, rather than wading through all the various chapters, can get an overview
of possible sample preparation methods that are most applicable to gases, liquids, suspensions, gels and solid
materials. So the reader can get directly to the sample prep methodology that may suit his/her particular sample. In
subsequent Chapters, rather than repeating all the information, I refer back to these tables. The book is organized by
the flow of sample preparation process (sampling, transport, storage all the way up to sample filtration and, in some
cases, sample introduction).
The Chapters are further organized by sample types so that gaseous samples are first (Chapter 6) followed by liquid
samples (or samples put into a liquid form) (Chapters 7-12), solid samples (Chapter 14), biological samples (nucleic
acids and proteins)(Chapter 15), biological fluids/tissues (Chapter 16) and then special topics such as sample prep for
mass spectrometry, membrane applications, chemical scavengers, and derivatizations. Finally, Chapter 21 covers a
new concept of “Just Enough” sample preparation that seems to be today’s trend relying heavily on the increased use
of hyphenated-chromatography/tandem mass spectrometry techniques. To aid the novice (and maybe some of the
experts!) on the terminology associated with sample prep, the final Chapter 22 includes a Glossary.
I wish you good reading and hope that the material within provides you with a good foundation on how to best approach
your sample preparation challenges.
Ronald E. Majors, Wilmington, DE
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Acknowledgements
First and foremost, I would like to thank my wife, Carol, for giving up part of our retirement time allowing me to work
on this book and for her support during this process. I would also like to thank Helen Stimson, my boss at Agilent, for
allowing me to work ½ time combining my pending retirement with my passion for science, especially sample
preparation technologies. I would also like to thank the three Agilent Marcom ladies (Christine Cox, Anne Batchelor,
and, especially Nicole Goodman who managed the bulk of the effort) for helping me get this volume to press. Special
thanks go to Agilent colleagues: to Edward Elgart who proofread the entire book, to Gina Goggins for her helpful
suggestions and locating some of the figures to use as examples and to Dennis Blevins who gave excellent detailed
feedback on the technical aspects of most of the chapters. In addition, I would like to thank Agilent sample prep and
chromatography colleagues who read specific chapters and gave their feedback. This feedback helped to shore up the
usefulness of the information and with helpful suggestions on where improvements could be made: Trisa Robarge,
Nigel Simpson, Christophe Deckers, Bill Wilson, Jared Bushey, Limian Zheo, Michael Woodman, Sue Cohen, Tina
Chambers, Jennifer Massi, Derick Lucas and Bruce Richter and finally to Vanita Marshall who kept track of and contact
with all of the reviewers.
Last but not least, I would like to thank LCGC No. Americafor allowing me to use some of the published information
and artwork used in various articles over the years from my monthly columns, Column Watch and Sample
Preparation Perspectives.
Ronald E. Majors, Wilmington, DE
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Chapter 1
Introduction
The major stages of an analytical process are depicted in Figure 1.1. The proper choice of the measurement technique is only one
step in the development of a successful application. All of the steps leading up to the measurement are as important. The sampling
and sample preparation process begins at the point of collection and extends to the measurement step. The proper collection of
sample during the sampling process (called primary sampling) is the initial contact with the sample and it is imperative that this
sample represents the entire lot being investigated. The storage, preservation and transport of this representative sample from the
point of collection to the analytical laboratory must occur without any changes in the physical and chemical makeup of the original
sample. The proper selection of the laboratory sample itself (termed secondary sampling) represents another challenge since the
final sample used for analysis may be a tiny fraction of the original collected sample, yet serves as a subset. Finally, the sample
preparation methodology necessary to convert the sample into a form suitable for the measurement step must also be performed
without loss or any unplanned modification of the secondary sample. All of these pre-analysis steps can have a greater effect in the
overall accuracy and reliability of the results than the measurement itself.
Figure 1.1
Sample Analysis Workflow Diagram
Sample analysis
Sample preparation
1%
Sample transport MeOH
Data handling
Archiving
Sample collection/sampling
Report generation
Information to customer
1
Sample preparation is an essential part of chromatographic and spectroscopic analyses. The process is intended to provide a
representative, reproducible, and homogenous solution that is suitable for injection into the column for chromatographic analysis,
or into an ICP-MS/atomic adsorption source, or into a cuvette or NMR tube for further characterization. The aim of sample
preparation is to provide a sample aliquot that (a) is relatively free of interferences, (b) will not damage the column or instrument
and (c) is compatible with the intended analytical method. In chromatography, the sample solvent should dissolve in the HPLC
mobile phase or be injectable into a GC column without affecting sample retention or resolution, the stationary phase itself, and
without interfering with detection. It is further desirable to concentrate the analytes and/or derivatize them for improved detection
or better separation. In spectroscopy, the sample solvent should be free of particulates, compatible with the spectroscopic source,
and be of the appropriate viscosity to flow into a nebulizer for on-line methods. Sometimes, depending on spectroscopic sensitivity,
preconcentration is needed and chromatography or liquid-liquid extraction is sometimes used prior to introduction of the sample
into the instrument.
Although many of the sample preparation protocols used in chromatography and spectroscopy are similar, it is beyond the scope of
this book to address the various differences between sample preparation procedures for these diverse methods. Therefore, we will
limit the topics in this handbook to the popular sample preparation methods for chromatographic analysis with emphasis on Liquid
Chromatography (LC)/High Performance LC (HPLC)/Ultra HPLC (UHPLC), and Gas Chromatography (GC).
A more detailed depiction of the various operations in the analytical cycle is summarized in Table 1.1. If particular attention is not paid
to all of these operations, sample integrity may be sacrificed and the analysis data affected, compromised, or rendered invalid. Steps
1-5, which include 1) sample collection, 2) storage and preservation, 3) sample transport, 4) preliminary processing and laboratory
sampling, and 5) weighing or dilution, all form an important part of sample preparation. Although these steps in the chromatographic
assay can have a critical effect on the accuracy, precision, and convenience of the final method, space limitations preclude us from
addressing all of these areas in detail. Only steps 1 and 4 (sample collection and preliminary sample processing) will be briefly
explained here. See References 1-4 for an explanation of steps 2, 3, and 5. The bulk of this book will be devoted mainly to Steps
6-9 of Table 1.1, which encompasses what is usually meant by sample pre-treatment or sample preparation (“sample prep”).
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Table 1.1
Sample Pre-treatment Options
Step Option Comment
1 Sample collection Obtain representative sample using statistically valid processes.
2 Sample storage and preservation Use appropriate inert, tightly-sealed containers; be especially careful with volatile,
unstable, or reactive materials; stabilize samples, if necessary; biological samples may
require refrigeration or freezing.
3 Sample transport The act of transporting the sample from the point of collection to the laboratory can be an
important step. Transportation conditions should maintain its integrity, samples should not
have rough handling, be dropped, or be allowed to be exposed to the elements; the
timing may be important for samples – undue delays may cause sample degradation
as in step 2 above.
4 Preliminary sample processing Sample must be in form for more efficient sample pre-treatment (e.g. drying, sieving,
grinding, etc.); finer dispersed samples are easier to obtain representative sample and
to dissolve or extract.
5 Weighing or volumetric dilution Take necessary precautions for reactive, unstable, or biological materials; for dilution,
use calibrated volumetric glassware.
6 Alternative sample processing methods Solvent exchange, desalting, evaporation, freeze drying, etc.
7 Removal of particulates Filtration, centrifugation, solid phase extraction.
8 Sample extraction Method for liquid samples (Table 2.4) and solid samples (Tables 2.2 and 2.3)
9 Derivatization Mainly to enhance analyte detection; sometimes used to improve separation, extra step
in analytical cycle adds time, complexity, and potential loss of sample (See Chapter 20).
Whereas GC and HPLC are predominantly automated procedures, sample pre-treatment is often performed manually. As a result,
sample pre-treatment can require more time for method development and routine analysis than is needed for the separation and
data analysis (see Figure 1.2). Sample pre-treatment may include a large number of methodologies, as well as multiple
operational steps, and can therefore be a challenging part of chromatographic method development.
3
Figure 1.2
Time Spent on Typical Chromatographic Analysis
Data management (27%)
Collection (6%)
Analysis (6%)
Sample processing (61%)
Data taken from Agilent Technologies survey
Finally, method precision and accuracy is frequently determined by the sample pre-treatment procedure (see Figure 1.3), including
operations such as weighing and dilution. For all of these reasons, the development of a sample pre-treatment procedure deserves
careful, advance planning.
Figure 1.3
Sources of Error Generated During Chromatographic Analysis
Contamination (4%) Sample introduction (6%)
Chromatography (7%)
Columns (11%)
Integration (6%)
Operator (19%)
Instrument (8%)
Calibration (9%)
Sample processing (30%)
Data taken from Agilent Technologies survey
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